Embryonic stem (ES) cell research offers unprecedented potential for understanding fundamental developmental processes, such as lineage differentiation. Embryonic stem cell lines are derived from early embryos and are characterized by their ability to self-renew, that is, to be maintained indefinitely in a proliferative and undifferentiated state in culture. ES cells are also pluripotent, meaning they retain the capacity to differentiate into the three embryonic lineages: ectoderm, mesoderm and endoderm plus all of their derivatives (Chambers I., 2004). The recent development of reprogramming technologies now allows ES-like stem cells to be generated from somatic cells, such as fibroblasts. Introduction into somatic cells of a small set of specific transcription factors—Oct4, Sox2, c-Myc, and Klf4 in the mouse (Takahashi and Yamanaka, 2006) and human (Park et al., 2008b; Takahashi et al., 2007), or Oct4, Sox2, Nanog and Lin28 in human (Yu et al., 2007)—can reprogram various differentiated cell types to an ES-like stem cell state (induced pluripotent stem cells or iPS). This strategy now allows the generation of ES-like cell lines from individual patients and, thus, offers the possibility to create highly relevant in vitro models of human genetic diseases. Such reprogrammed cell lines have already been generated from patients with a variety of diseases, such as Duchenne Muscular Dystrophy or Amyotrophic lateral sclerosis (ALS) and differentiation of the reprogrammed cells into the deficient tissue has been achieved for iPS cells from patients affected with several diseases such as ALS, thus, demonstrating the feasibility of the approach (Dimos et al., 2008; Park et al., 2008a).
Whereas some lineages such as cardiac myocytes or neurons are easily generated in vitro from ES cells, differentiating paraxial mesoderm derivatives such as skeletal muscle, dermis, cartilage or bone from ES or iPS cells has proven to be challenging. Given the promises offered by cellular replacement therapy for the cure of some muscular degenerative diseases or for orthopaedic surgery, the development of protocols for production of precursors of muscle and skeletal lineages is of key importance. In the embryo, the muscles, the dorsal dermis and the axial skeleton of the body derive from the paraxial mesoderm and more specifically from multipotent precursors forming the presomitic mesoderm (PSM). These precursors are characterized by expression of the genes Brachyury (T), Tbx6 and Mesogenin1 (Msgn1) (Chapman et al., 1996; Yoon and Wold, 2000) and they mostly differentiate into skeletal muscles, dermis, skeletal lineages, as well as in a variety of other derivatives including adipocytes and endothelial cells. In the mouse embryo, Rspo3 (also called Cristin1, Thsd2) is strongly expressed in the PSM and somites, as well as later in condensing mesenchymal cells, (Kazanskaya et al., 2004; Nam et al., 2007). R-spondins (Rspo1 to 4 genes) are secreted molecules containing a thrombospondin domain, that can activate canonical Wnt signaling and Beta-Catenin, via the Fzd/LRP/Lgr4/Lgr5 co-receptors complex (Carmon et al., 2011; de Lau et al., 2011; Kim et al., 2008; Nam et al., 2006), but they were also shown to bind Syndecan4 and induce Wnt/PCP signaling (Ohkawara et al., 2011). Interestingly, biochemical assays show that Rspo2 and 3 are more potent to activate Wnt signaling than Rspo1 and 4 (Kim et al., 2008). R-spondins have also been shown to be implicated in bone formation and chondrogenesis (Hankenson et al., 2010; Jin et al., 2011; Ohkawara et al., 2011), myogenesis (Han et al., 2011; Kazanskaya et al., 2004) and angiogenesis (Kazanskaya et al., 2008).
Bone Morphogenetic Proteins (BMPs) are secreted molecules of the TGFbeta superfamily that can dimerize and activate BMP signaling and bind to a receptor complex constituted of BMP receptor type I and type II (BMPR-I and -II). More precisely, BMPR-I can consist of Activin receptor-like kinase (ALK)-2/3 and 6 (also known as ActR-IA, BMPR-IA and BMPR-IB respectively). Similarly, BMPR-II can consist of BMPR-II, ActR-IIA and ActR-IIB. The BMP receptor complex is formed by an heterotetrameric complex of two BMPR-I and two BMPR-II. The BMP receptor contains an intracytoplasmic serine/threonine kinase domain which allows the phosphorylation of Smad 1/5/8 upon binding of the BMP dimer. Phosphorylated Smad1/5/8 then associate to Smad4 and shuttle to the nucleus to activate target genes, which include the inhibitor of DNA binding (Id) 1/2/3 genes [Hollnagel A et al., 1999]. Importantly, numerous BMP/TGFβ secreted agonists and antagonists have been described to regulate and fine-tune BMP signaling during development. Most notably noggin, chordin, follistatin and gremlin block BMP signaling by sequestrating secreted BMP, preventing its binding to the receptor. BMP ligands (prominently BMP2, 4 and 7), BMP receptors, Smads, Co-Smads and BMP agonists/antagonists have been implicated in mesoderm specification and organogenesis during development [Derynck Rik, 2008; Reshef R. et al, Gen Dev 1998; Wijgerde M. et al, 2005; McMahon J A et al, 1998; Stafford D A et al, 2011; Pourquié O. et al, 1996 and Tonegawa A. et al, 1997].
Differentiation of ES cells into paraxial mesoderm and its derivatives is highly inefficient in vitro. Limited spontaneous skeletal muscle differentiation has been described following culture of mouse embryoid bodies and DMSO treatment (Dinsmore et al., 1996; Rohwedel et al., 1994), or Retinoic acid treatment (Kennedy et al., 2009). Two distinct strategies to differentiate mouse and human ES cells in vitro to the muscle lineage have been reported. The first one involves the sorting of precursors using surface markers. For instance, Studer's group reported the isolation of human ES cells-derived CD73+ mesenchymal precursors and their subsequent differentiation into skeletal muscle following a culture period in serum containing medium (Barberi et al., 2007). The antibody against satellite cells SM/C-2.6 was also used to isolate myogenic cells differentiated from mouse ES and iPS cells (Fukada et al., 2004; Mizuno et al., 2010). Finally, mesoderm precursors differentiated from mouse ES cells were also isolated based on their expression of other surface markers such as the Platelet derived growth factor receptor alpha (PDGFRa) or Vascular endothelial growth factor receptor 2 (VEGFR2) ((Sakurai et al., 2009; Sakurai et al., 2008) Sakurai H. et al., 2006; Takebe A. et al, 2006). Whether this combination of markers is strictly specific for paraxial mesoderm precursors has however not been demonstrated. The second strategy is based on forced expression of the transcription factors Pax3 or MyoD, or of the secreted factor Insulin Growth Factor 2 (IGF-2) in mouse ES cells (Darabi et al., 2008; Darabi et al., 2011; Dekel et al., 1992; Prelle et al., 2000; Shani et al., 1992). However, these strategies show either limited efficiency or require introduction of exogenous DNA in the ES cells which is a major hurdle for the development of safe cell therapies and the differentiated cells often show limited proliferation and engraftment potential.
Therefore, there is a need to develop better ES and iPS cell differentiation strategies to produce muscle cells and paraxial mesoderm derived lineages for the development of applications in regenerative medicine.
The present invention fulfils this need by providing a method for preparing multipotent progenitor cell lines expressing markers of the paraxial mesoderm progenitors and referred to as induced Paraxial Mesoderm progenitor cells or iPAM to distinguish them from the natural embryo Paraxial Mesoderm progenitor cells. Like their in vivo counterpart, the iPAM cells are capable of giving rise to cell lineages of the muscular, skeletal (bone and cartilage), dermal tissue, and derivatives such as adipocytes and endothelium. The inventors have shown that embryonic stem cells or pluripotent reprogrammed cells (iPS) can be differentiated into induced Paraxial Mesoderm progenitor (iPAM) cells using a limited number of factors. In particular, the inventors have made the surprising finding that it is possible to efficiently obtain induced Paraxial Mesoderm progenitor (iPAM) cells by treatment with specific factors, without any genetic modification of the target cells. They have shown that the obtained induced Paraxial Mesoderm progenitor (iPAM) cells exhibit characteristics of endogenous Paraxial mesoderm progenitor cells. To the applicant's knowledge, the invention is the first description of a method for obtaining unlimited amounts of cells suitable for use as progenitor cells for regenerating either muscle, skeletal, adipose or dermal tissues and paraxial mesoderm derived endothelium. Therefore the invention is highly useful in particular in regenerative medicine.